Trypanosoma cruzi is a protozoan parasite that causes Chagas' disease, which affects several million people and constitutes a major health problem in South and Central America. No effective drug is available against this disease and no vaccine has been developed, even though basic research has been carried out into the biology of T. cruzi for decades. The large amount of useful data produced in the last years within various genome projects have demonstrated the power of this approach for gene finding and genome characterization. Therefore, in the last three years, pilot genome mapping and sequencing projects have been carried out to increase the efficiency of gene finding to identify potential drug targets and to create a knowledge base for future genome efforts. These projects have included initial karyotyping, mapping of several chromosomes, EST sequencing and sequencing of chromosome 3. The latter project as well as mapping and EST sequencing has been carried out in this laboratory. In order to take advantage of the knowledge of the genome organization of T. cruzi that has been accumulated, we here propose, together with groups at TIGR (Najib El-Sayed) and SBRI (Ken Stuart), to initiate a large effort to characterize the T. cruzi genome. The strain of choice will be the CL Brener reference strain that has been used in the initial projects. The haploid genome size of this organism is approximately 43.5 Mb. The goals of the project are: (1.) to characterize the karyotype of this strain by hybridization of 1,000 specific markers to separated chromosomes, which will result in a linkage map with a marker every 40-50 kb. (2.) to carry out end-sequencing from genomic BAC- clones in order to establish a physical map and for gene discovery. (3.) sequencing of chromosomes in T. cruzi CL Brener using clones selected on the basis of the marker and end-sequence information obtained from all three laboratories in (2.) Large-insert clone end sequencing and karyotyping will be carried out in the first year of the proposal and years 2 and 3 will be dedicated to chromosome-by-chromosome sequencing. We propose to carry out the karyotyping project as well as participate in the sequencing effort by carrying out end-sequencing from 7,000 BAC clone and complete sequencing of 10-15 Mb of genomic DNA in years 2 and 3. This project will result in a wealth of data on potential targets of drugs and vaccines and will, together with data from other parasite genome projects, be an extremely valuable resource for parasitologists world wide. We foresee that this project will bring about a plethora of investigations into gene function, gene expression, evolutionary and functional studies based on computational biology methods, and testing of potential drug targets.